In a luminescent reaction with luciferin/luciferase in vitro, conventionally, the luminescence pattern thereof is observed in a flash state. Therefore, it is impossible to measure the luminescent reaction accurately without using a device having a specific mechanism of injecting a reagent. However, K. V. Wood et al. has established a luminescence system having a relatively long half-life period of luminescence as long as 5 minutes. (Wood, K. V. Recent advantage and prospects for beetle luciferase as genetic reporters. In: Bioluminescence and Chemiluminescence current status. Proceeding of the VIth International Symposium on Bioluminescence and Chemiluminescence, Cambridge, September 1990. P.543. Ed. by P. Stanley and L. J. Kricka.). Owing to this, it has become possible to accurately measure a luminescence unit in a luciferin/luciferase system with a luminometer or a liquid scintillation counter which does not have an automatic reagent-injecting device. So, a reporter assay method wherein luciferase generated as a reporter in a culture cell is measured at a high sensitivity has been widely used. However, when the reporter assay method is used in a High Throughput screening for developing a medication, the luminescence half-life period of 5 minutes is insufficient. For example, for measuring many samples by using 96 wells, luminescent reagents must be added several times and measurement must be carried out each time. Like this, restrictions on operation and measurement exist.
Further, luciferin contained as a luminescent substrate in a luminescent reagent is apt to be oxidized in a solution during storage, to form an oxyluciferine. It is well-known that the oxyluciferin inhibits the enzyme reaction of a luciferase. Conventional luminescent reagents containing luciferin as a luminescent substrate have a big problem that a luminescent activity deteriorates due to the oxidation of the luciferin. This problem prevents the further expansion of uses for the assay using luciferase as a reporter. Therefore, the development of a new stabilization method is expected.
It is an object of the present invention to provide a luminescence method for luciferin/luciferase system, which extends the half-life period of a luminescent reaction, and a luciferase assay reagent.
It is an another object of the present invention to a luminescence method for luciferin/luciferase system, which prevents the deterioration of a luminescent activity and gives a stable luminescence reaction, and a Luciferase assay reagent.